2010 Annual Report
1a.Objectives (from AD-416)
This Project is comprosed of four objectives designed to address key areas of research related to malignant catarrhal fever (MCF) and its causative viruses, particularly ovine herpesvirus 2 (OvHV-2). Discovery of new viruses in the MCF virus group necessitates extension of current nucleic acid based diagnostic tests with an emphasis on developing rapid and reliable assays for use by veterinary diagnosticians. In addition this project seeks to define basic virus-host interactions at the molecular level in order to identify how OvHV-2 causes disease so that control strategies, including immunological based methods such as vaccination, can be developed to protect clinically susceptible ruminants. These four objectives include.
1)extend nucleic acid based diagnostic tests to include newly discovered members in the MCF virus group;.
2)define host-virus interactions in sheep and develop an in vitro propagation system for OvHV-2;.
3)define OvHV-2 gene expression within MCF lesions; and.
4)develop an immunological control strategy for MCF in clinically susceptible ruminants.
1b.Approach (from AD-416)
Extend current nucleic acid-based tests for clinical diagnosis of MCF by validating the recently developed real-time PCR using a large set of defined field samples from animals with clinical MCF and by developing DNA microarray-based PCR for detection and differentiation of MCF group viruses. MCF pathogenesis will be studied in three phases:.
1)characterize lesion development in bison during preclinical and clinical stages;.
2)determine OvHV-2 genes that are highly expressed during preclinical and clinical stages using a gene expression microarray containing all 73 OvHV-2 open reading frames; and.
3)define the role of OvHV-2 gene products in MCF lesion development by developing an infectious OvHV-2 bacterial artificial chromosome (BAC) clone and testing the pathogenicity of infectious OvHV-2 BAC clones in bison, with the deletion of genes associated with lesion development. In developing an immunological control strategy for MCF in clinically susceptible ruminants, we will first characterize bison MHC class I and class II haplotypes and determine any association between MHC specific alleles and MCF resistance/susceptibility. For analysis of immune responses to OvHV-2 and development of vaccines for protection of animals from MCF losses, we will determine if animals that survive initial low dose infection with OvHV-2 are resistant to clinical MCF after a subsequent high dose challenge using our recently established animal model, bison. We will also determine which immune response dominates in the animals that survive the challenge, and finally evaluate vaccine candidates for protection against MCF in clinically susceptible ruminants. Replacing 5348-32000-018-00D October, 2006.
The scientists at the Animal Disease Research Unit, in collaboration with Washington State University and the University of Wyoming, defined virus-host interactions in bison, a species highly susceptible to malignant catarrhal fever (MCF) and compared it with sheep, a natural carrier. The study revealed that the early viral replication that occurred in bison lung was similar to that in sheep; however, there was no significant increase in immune response gene transcriptions in the bison lung as there was in sheep. Subsequent systemic dissemination of OvHV-2 was followed by lytic gene expression in nearly all tissues preceding onset of clinical signs, which was significantly different from what happened in sheep, which was followed by dissemination of latently infected cells. We further determined that the expression of an OvHV-2 lytic gene (ORF25), a gene marker for viral replication, was significantly correlated with lesion severity in bison. Overall, these data suggest that OvHV-2 may change cell tropism during initial replication in lung of both carrier and clinically susceptible hosts, and the ultimate differences between subclinical infection and disease in these species may depend on their immune functions as modulated by the virus. We also detected OvHV-2 capsid proteins in tissue lesions from bison with experimentally OvHV-2-induced-MCF, using dual-stain fluorescent technology. In this study, we discovered an unexpected cell population, activated fibroblast cells, with expression of cytoplasmic staining of viral proteins. In order to understand the difference in pathogenesis between OvHV-2 and AlHV-1, the wildebeest-associated MCF virus, we launched a study to characterize lymphoproliferation and viral gene expression in experimentally infected rabbits with OvHV-2-induced MCF, while confirming that rabbits are an excellent model for OvHV-2 research. The results showed that there were significant differences in pathogenesis between the two viruses, although they cause similar clinical signs and pathological lesions. Our study revealed that OvHV-2 induced pan-lymphoproliferation associated with lytic gene expression in the clinical stage, as compared to a previous study which indicated only CD8+ cell proliferation with latency-associated gene expression. These studies suggest that the pathogenesis of OvHV-2 is different from previously proposed hypothesis that it is associated with viral latency and also from the pathogenesis of AlHV-1. This information is critical for the development of immunological strategies to prevent the disease. In addition, we have tried several cell lines, including primary sheep lung epithelial cells derived from newborn lambs, to determine if OvHV-2 can be grown in cell culture. Despite many attempts, we failed to culture OvHV-2, possibly due to changes in cells resulting from in vitro culture, even though these cells were confirmed to support OvHV-2 replication in sheep. To pave the foundation for the next 5-year research plan we initiated sequencing and genome annotation of AlHV-2, a non-pathogenic MCF virus from African antelope, which will potentially be used as a vaccine backbone for MCF.
Determined that the expression of OvHV-2 lytic genes is correlated with lesion severity in bison. How OvHV-2 causes malignant catarrhal fever (MCF) has been a matter of debate; however, it has been widely accepted that viral latency (viral genomes maintained without replication in infected cells) is the main feature leading to the disease. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at Washington State University, Pullman, Washington, and the University of Wyoming, Laramie, Wyoming, determined viral replication status and characterized lesion development in various tissues at preclinical and clinical stages of MCF. Results indicated that the levels of a viral gene coding for a structural protein expressed during virus replication were significantly correlated with lesion severity in tissues, suggesting that the development of lesions is predominantly associated with viral replication, rather than with latency. This is additional evidence to suggest that the disease mechanism for OvHV-2 is different from previously proposed hypotheses related to latency, and this information is critical for development of immunological strategies to prevent the disease.
Developed an OvHV-2-induced malignant catarrhal fever (MCF) rabbit model and determined that the mechanism of OvHV-2-induced disease in rabbits differed from that of AlHV-1, the wildebeest-associated MCF virus. Development of laboratory animal models using the nasal route of infection to mimic natural virus transmission has been challenged in the past and questions have been raised as to whether the two viruses OvHV-2 and AlHV-1 use different mechanisms to induce the disease, although the clinical signs and pathological lesions caused by the two viruses are similar. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at Washington State University, Pullman, Washington, the University of Wyoming, Laramie, Wyoming, and the University of Liège, Belgium, developed an OvHV-2-induced MCF model using rabbits and discovered that the mechanism of OvHV-2-induced disease in rabbits was in fact different from that of AlHV-1-induced MCF in rabbits. The study successfully induced the disease in 100% of the rabbits and also revealed that OvHV-2 induced all T and B lymphocyte subsets to proliferate in association with a virus lytic gene expression profile in the clinical stage, as compared to a previous study with AlHV-1 which indicated only a subset of T lymphocytes (called CD8+ cells) proliferated with latency-associated gene expression. This is the first study to document that OvHV-2 is different from AlHV-1 in induction of disease in rabbits. The establishment of a rabbit model for OvHV-2 allows us to perform animal experimental studies with the goal of vaccine development and better control of virus transmission and disease before conducting the research in bison.
Identified a viral mechanism resulting in development of MCF in animals. There is little information about how malignant catarrhal fever (MCF) develops in animals and there is a huge difference in disease susceptibility among species, including sheep, cattle and bison. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at Washington State University, Pullman, Washington, and the University of Wyoming, Laramie, Wyoming, determined the kinetics of OvHV-2 replication and evaluated host immune response gene expression following infection in bison. We determined when and where viral replication and dissemination occur in bison; we found that the virus exhibited uncontrolled replication in the clinical stage and that the host showed little immune response during infection, suggesting that the host immune system was suppressed by the virus leading to clinical disease. This is the first evidence suggesting that the disease mechanism for OvHV-2 is different from previously proposed hypotheses related to latency; this information is critical for the development of immunological strategies to prevent the disease.
5.Significant Activities that Support Special Target Populations
Research on epidemiology, transmission, and control strategies has a significant impact on MCF control programs for American bison producers, game farms, wild stocks, zoological collections, and state fair management.
Nelson, D.D., Davis, W.C., Brown, W.C., Li, H., O'Toole, D., Oaks, J.L. 2010. CD8+/perforin+/WC1- gammadelta T cells, CD8+ alphabeta T cells, infiltrate vasculitis lesions of American bison (Bison bison) with experimental sheep-associated malignant catarrhal fever. Veterinary Immunology and Immunopathology. 136(3-4):284-291.
Taus, N.S., Schneider, D.A., Oaks, J.L., Yan, H., Gailbreath, K.L., Knowles Jr, D.P., Li, H. 2010. Sheep (Ovis aries) airway epithelial cells support ovine herpesvirus 2 lytic replication in vivo. Veterinary Microbiology. 145(1-2):47-53.
Hamir, A.N., Palmer, M., Li, H., Stasko, J., Rogers, D.G. 2009. Spontaneous Idiopathic Arteritis of the Testicular Artery in Raccoons (Procyon lotor). Veterinary Pathology. 46(6):1129-1132.
Moore, D.A., Kohrs, P., Baszler, T., Faux, C., Sathre, P., Wenz, J.R., Eldridge, L., Li, H. 2010. Outbreak of malignant catarrhal fever among cattle associated with a state livestock exhibition. Journal of the American Veterinary Medical Association. 237(1):87-92.
Gailbreath, K.L., O'Toole, D., Taus, N.S., Knowles Jr, D.P., Oaks, J.L., Li, H. 2010. Experimental nebulization of American bison (Bison bison) with low doses of ovine herpesvirus 2 from sheep nasal secretions. Veterinary Microbiology. 143(2-4):389-393.
Cunha, C.W., Otto, L., Taus, N.S., Knowles Jr, D.P., Li, H. 2009. Development of a Multiplex Real-Time PCR for Detection and Differentiation of Malignant Catarrhal Fever Viruses in Clinical Samples. Journal of Clinical Microbiology. 47(8):2586-2589.